In this competitive renewal application, we will investigate specific aspects of the biology of locally advanced breast caners (LABCs) that were uncovered by our initial set of studies. Our previous specific aims were to (1) measure the response of LABC to neo-adjuvant chemotherapy using [Tc-99m]-sestamibi (MIBI) and [F-181]-fluorodeoxyglucose (FDG) PET and (2) to characterize the physiology of tumor response using MIBI, FDG, and [0-15]-water with the goal of identifying mechanisms of resistance. Our studies established serial MIBI imaging as an accurate toll for measuring response and indicated the accuracy of FDG PET for initial LABC staging and determining the extent of viable tumor post-therapy. In addition, we found a very striding set of findings regarding the physiology of responding tumors: (1) Tumors with a high rate of glycolysis (high FDG uptake) pre-therapy responded poorly to chemotherapy. (2) Patients who ultimately achieved a complete macroscopic response to therapy (CP) had a drop in tumor blood flow after two months of therapy, while all others had no change or an increase in blood flow; this pattern did not hold for the change in FDG uptake. Our studies also suggested that MIBI uptake is largely determined by blood flow and that neither pre-therapy MIBI uptake nor washout is a reliable predictor of chemotherapy resistance. Recent analysis has shown that the ratio of pre-therapy glycolysis to blood flow and the level of residual blood flow and MIBI uptake post-therapy predict disease-free survival. We now focus on mechanisms of tumor response and treatment resistance suggested by our first set of studies. By comparing PET measurements of tumor physiology to response and to in vitro assays of tumor biologic properties, we will investigate (1) the mechanisms underlying the apparent chemotherapy resistance of highly glycolytic LABC, in particular tumor hypoxia, (2) methods to measure response early in the course of therapy using PET cell proliferation tracers, and (3) the kinetic properties of MIBI using the positron-emitter [Tc-94m]-sestamibi to more precisely determine the effect of blood flow and efflux pump expression on MIBI uptake and washout. Our studies will increase the understanding of resistance and response to chemotherapy in advanced breast cancer and generate new approaches to individualizing patient treatment to optimize response. In this amended application, we focus on recent progress that has strengthened our preliminary data and further supports our proposed investigations of the biology of advanced breast cancer and its relationship to therapeutic response.